Project Summary There is an urgent and unmet need for the development of safe and effective therapeutics against serious human pathogens such as Kaposi's sarcoma herpesvirus (KSHV), which causes significant morbidity and mortality in immune-compromised individuals and remains a clinical challenge. Currently, no FDA approved therapeutics or vaccines are available for KSHV infection. Given that KSHV and other human herpesviruses hijack host proteins and pathways to complete their replication cycles and spread from cell to cell, strategies targeting these pathways and mechanisms will provide broad-spectrum genotype coverage and a high barrier to drug resistance. Herpesviruses including KSHV have long been known to exploit the COPII-mediated secretory pathway for their maturation. However, the mechanisms governing this process remain less understood. This project will fill this gap, capitalizing on our recent discovery that the host nucleoside diphosphates kinase NM23-H2, an important regulator of COPII vesicle transport, is exploited by KSHV for their virion morphogenesis and egress. We found that viral Bcl-2 of KSHV (ks-Bcl-2) directly interacted with, stabilized, and activated NM23-H2 during lytic phase of KSHV. Loss of NM23-H2 or mutations in ks-Bcl-2 that abolished NM23-H2 interaction severely impaired virion production. We thus hypothesize that KSHV activates host protein NM23-H2 to exploit the COPII pathway for efficient virion assembly and release, unraveling a key checkpoint in virus lifecycle that can be targeted for new antiviral therapeutics. We now bring within this proposal a collaboration of experts in KSHV biology and in design of small-molecule inhibitors to identify cellular pathway responsible for virion production of KSHV and develop a new strategy to dampen virus transmission. To achieve this goal, we propose two specific aims, including (1) defining the molecular mechanism by which KSHV ks-Bcl-2 targets NM23-H2 to activate Sar1-mediated COPII transport for efficient virion assembly; and (2) targeting ks-Bcl-2-NM23-H2-mediated COPII mechanism to block KSHV propagation. These aims will be addressed using multidisciplinary approaches that integrate state-of-the-art genetic, biochemistry, live-cell imaging, and physiological assays. Together, we anticipate that our studies will identify host genes/pathways that function in virus assembly and egress, and provide compelling in vivo validation that targeting NM23-H2-dependent host mechanism can abrogate virus transmission within and between individuals.